Among the currently available techniques for high throughput proteomics, protein microarrays have the greatest prospects to revolutionize molecular diagnostics for early detection, diagnosis, treatment, prognosis and monitoring clinical response. However, protein microarrays have yet to reach their full potential as a research or clinical molecular diagnostics tool due to difficulties associated with their manufacture. Currently protein microarrays are manufactured by expressing & purifying thousands of proteins, which are then stored until they are printed using pin-spotters, a process flow with many inherent logistical problems. Furthermore, many proteins are unstable so these steps must all be maintained at cold temperature. Problems associated with pin spotters include: relatively slow printing speeds, poor spot morphology, pin biofouling issues, variable spot sizes, limited microarray densities and others. Thus, there are compelling needs for better and less expensive manufacturing methods for protein microarrays. In this grant we will combine two successful technologies to develop an innovative method for mass production of faster, better and cheaper protein microarrays. One technology is based on our advanced high speed piezoelectric pipettes to print arrays of cDNA templates and the other is to express proteins in situ directly on the microarray surface. Engineering Arts specializes in providing microarray production solutions based on its proprietary piezoelectric pipetting technology. Dr. LaBaer is the co-inventor of nucleic acid programmable protein arrays (NAPPA): the very first method to express proteins in situ directly in a microarray format. Engineering Arts will install one of its production-scale piezoelectric microarray machines (POC2) in Dr. LaBaer's Center for Personalized Diagnostics (CPD), Biodesign Institute, Arizona State University. We will develop tools, protocols and process controls required to manufacture production-scale, commercial-grade, high-density, customizable protein microarrays making them readily accessible to the broad proteomics research and clinical diagnostics communities. This grant directly addresses the call to develop a broadly applicable research tool that addresses a core technical challenge in proteomics. By making high quality protein microarrays more readily assessable, this grant will help unlock their true potential for research and clinical applications. This grant brings together world-class piezoelectric pipettes and electronics developed at Engineering Arts, over ten years experience in developing commercial automated production-scale piezoelectric microarraying manufacturing capabilities for high-density whole-genome gene expression microarrays; world class production-scale automation process manufacturing equipment from an established Singapore based semiconductor production equipment manufacturer, Dr. LaBaer's unique and patented NAPPA technology together in his CPD to develop, characterize and validate the next generation of commercial protein microarrays. PUBLIC HEALTH RELEVANCE: Nearly all diagnostics and therapeutics act through proteins, which are the working machines of biology. The study of proteins, both their activities and their dysfunction in disease, has been historically managed one- protein-at-a-time; however, this will be dramatically accelerated through the use of protein microarrays, which microscopically display thousands of functional proteins. This grant will develop technology to mass produce better and less expensive protein microarrays, making them more readily accessible to the broad research and health care communities.